The major goal of this project is to generate a comprehensive picture of cellular mechanisms regulating intracellular copper levels through copper delivery to the secretory pathway and copper export from the cell. The project focuses on the analysis of copper-transporting ATPases ATP7A and ATP7B, which play an essential role in this regulation. In humans, mutations in these transporters cause severe metabolic disorders, Menkes disease and Wilson's disease, respectively. Specific Aim 1 outlines experiments dissecting the mechanism of copper release from Cu-ATPases. The hypothesis will be tested that the first luminal loop of the Cu-ATPases stimulates copper release from the transporters by sequestering copper at the previously unidentified metalbinding sites. The metal-binding properties of the loop region will be characterized, its structure will be determined by NMR, and the effect of mutations within the first loop on the function of Cu-ATPases will be examined. Specific Aim 2 will determine the role of luminal copper acceptor proteins in transport activity and trafficking of Cu-ATPases from the secretory pathway. The experiments under Specific Aim 3 will investigate specific functions of ATP7A and ATP7B in balancing copper concentration in kidney. Both Wilson's disease and Menkes disease are associated with copper accumulation in renal proximal tubules suggesting distinct and nonoverlapping roles of ATP7A and ATP7B in renal copper homeostasis. This hypothesis will be tested by determining the effect of Cu-ATPases inactivation on total cellular copper content and on copper efflux from the basolateral and apical membranes of polarized renal cells. The effect of small molecular weight compound, which is highly elevated in the serum and urine of Atp7b-/- mice, on copper uptake and localization of ATP7A will be determined. The studies will produce novel mechanistic information necessary for better understanding of human copper homeostasis and better treatment of disorders associated with copper misbalance.